1. Field of Invention
The present invention relates to improvements in methods and apparatus for differentiating and enumerating the five major sub-populations of leukocytes (white blood cells) in a human blood sample. The invention is particularly useful for differentiating and enumerating the basophil sub-population of leukocytes in a blood sample.
2. Discussion of the Prior Art
Conventional hematology instruments are capable of differentiating and enumerating the five major sub-populations of leukocytes (white cells) in a human blood sample, namely, the lymphocyte, monocyte, neutrophil, eosinophil and basophil sub-populations. Such instruments commonly operate by first lysing the erythrocytes (red cells) in a whole blood sample, and then causing the remaining leukocytes in the sample to flow, substantially one-at-a-time, through a narrow aperture or cell interrogation zone while subjecting each cell to a combination of electrical and light energy. While passing through the interrogation zone, a combination of measurements are made to determine each leukocyte's unique characteristics in terms of light scatter, Coulter DC volume, radio frequency (RF) electrical conductivity, polarization, and/or fluorescence. Of the five sub-populations of leukocytes, the basophils present a particularly difficult challenge to differentiate owing to (1) their relatively low number (compared to the number of other leukocytes in a normal human blood sample), and (2) their similarity to monocytes and lymphocytes in responding to electrical and light stimuli, whereby the basophils appear in a region of parameter space where monocytes and lymphocytes are also found. To further complicate matters, the basophils are often not readily distinguishable as a tight cluster in a scattergram in which two measured cell parameters are plotted against each other. This problem is discussed in the article by Terstappen et. al., Cytometry 9:39-43 (1988), where a scattergram obtained by comparing light scatter measurements at two different angular ranges, 1.degree.-2.6.degree. and 3.degree.-11.degree., is used to resolve the three major populations of white blood cells, namely, the lymphocytes, monocytes and granulocytes which, in this case, include the eosinophil and neutrophil sub-populations). In this scattergram, the basophils were not resolvable as an independent cluster although a general region or "gated" area was tentatively given where the basophils tend to appear. However, it was the conclusion of the authors of that article that this gated area was not sufficient to obtain a reliable basophil count.
Other investigators have taken different approaches to solving the problem of differentiating and enumerating basophils. For example, in U.S. Pat. No. 5,125,737, Rodriguez et. al., the disclosure of which is incorporated herein by reference, the problem of overlapping lymphocyte and basophil sub-populations is addressed by using, in addition to DC volume measurements and light scatter measurements within certain relatively broad angular ranges between 10 degrees and 70 degrees, an additional measurement parameter termed "opacity" is used. Opacity is defined as the ratio of a cell's DC impedance (volume) to its RF conductivity. While useful in resolving the basophils from other leukocytes, this approach requires additional circuitry for producing the requisite RF electrical field in the cell-interrogation zone, as well as circuitry for detecting changes in the RF current, as occasioned by the passage of cells through the zone. Owing to signal-to-noise issues, this approach is not as simple as it may sound.
Another approach for differentiating basophils, used in the. H*1 Hematology Analyzer manufactured by Technicon, Inc., employs a two-step chemical process for differentially lysing the other leukocyte sub-populations. Obviously, the time needed for two sequential chemical processes and the cost of additional reagents are disadvantageous. Still another approach is disclosed by HubI et al. [J. Clin. Lab. Anal. 10:177-183 (1996)] where basophils are identified by using double staining with fluorescence-labeled monoclonal antibodies. Other special methods, such as staining of heparin within the basophils at low pH and in the presence of lanthanum ions, have also been used [Gilbert et. al., Blood, 46:279-286 (1975)] to resolve basophils. As suggested, all of these prior art approaches are relatively complex and, ideally, should be simplified.
In addition to the above-noted problem of resolving basophils in a whole blood sample, the eosinophil sub-population of leukocytes also requires special attention in providing a 5-part differential analysis. In some measurement schemes, eosinophils tend to "look like" neutrophils (i.e., in parameter space). The above-noted Terstappen et al. article also discloses the use of orthogonal depolarized light scatter and orthogonal total light scatter intensities to resolve eosinophils from the neutrophils. This method is based on an observation that the refractile granules in the eosinophils tend to induce a greater depolarization of the scattered light in the orthogonal direction. Since this depolarization effect is stronger for the eosinophils than the neutrophils, a scattergram obtained by comparing depolarized orthogonal light scatter with total orthogonal light scatter intensity resolves the eosinophils as a cluster separate from the neutrophils. The method of Terstappen et al. has been used subsequently by Marshal to resolve the eosinophil population in whole blood, as disclosed in U.S. Pat. No. 5,510,267. However, it is generally known that the polarization effect of light scatter is more subtle than angular dependence of total light scatter intensity. Therefore, in general, the detection system required to measure depolarization must be more sensitive than that required for discerning angular variation of total light scatter intensity.